It is known to prepare useful polypeptides and proteins, for example enzymes, hormones, and (for use in e.g. vaccine preparation) toxins and other antigens, by cultivation of bacteria carrying plasmids with genes coding for the desired polypeptides or proteins. It is also known to construct plasmids containing desired genes by so-called recombinant DNA technique, which makes it possible to obtain, from the cultivated bacteria carrying such recombinant DNA plasmids, gene products which inherently are characteristic to other organisms than the bacteria used as host cells. In the preparation of recombinant DNA, a so-called cloning vector, that is, a plasmid which is able to replicate in the host bacterium, is combined with a DNA fragment containing a gene or genes coding for the desired product or products.
The recombinant DNA technique, in its most useful form, is based on the following principle:
DNA can be cut into pieces in a very specific way by restriction endonucleases. These pieces can then be joined to each other by DNA ligase. DNA cloning utilizes a plasmid vector that is a circular DNA molecule containing only one site for one or several restriction endonucleases. Treatment of such a vector with a restriction enzyme gives one species of a linear molecule. If this molecule is mixed with a DNA sample that is also treated with the same endonuclease, it is possible by ligation to obtain molecules composed of the vector to which a foreign DNA fragment has been fused. These plasmid molecules are called recombinant DNA. The vector with the foreign DNA can be transformed into a bacterial host cell, which means that it is taken up by and replicated in the bacterial host. Since the vector is able to replicate, the foreign DNA is also replicated.
If the foreign DNA is transcribed and translated in the bacterial host, the gene products of the foreign DNA are produced in the bacterial host. This production is in general proportional to the gene concentration which, on its side is proportional to the number of copies of the recombinant DNA plasmid molecules per cell. This means that in order to obtain large quantities of the desired gene products of the plasmid, a high number of copies of the plasmid per bacterial cell should be aimed at. It is known that some cloning vectors inherently replicate in a high copy number per bacterial cell, up to about 100. However, if the gene product produced by the foreign DNA combined with such cloning vector is one which is not well tolerated by the bacterial host, there may be difficulties in propagating a bacterial clone up to the desired production size culture because of inhibition exerted by the gene product. On the other hand, even a copy number of the order of about 20-100 will not always give rise to satisfactory yield of the desired gene product in the production culture. It is known that the number of copies of plasmids can be further increased by amplifying the inhibition of the protein synthesis, for example by addition of chloramphenicol, but as protein synthesis is necessary for preparing gene products of the cloned DNA, the amplified DNA will only be useful for formation of gene products thereof if the protein synthesis inhibiting component can be removed again, which is not always possible and often a complicated procedure.